Regulatory T cells (Treg) are fundamental components of the immune system, preventing the occurrence of many immune-related diseases, such as autoimmune diseases and allergies. Natural Foxp3+ Treg cells are selected in the thymus; it is believed that Treg cells are induced through interactions between their T cell receptor (TCR) and high affinity thymus ligands (agonists) in the presence of a permissive cytokine environment. The process is often referred to as agonist selection. Despite the importance of knowing how to induce Treg cells, a natural thymic ligand (agonist) that induces Treg cell development has never been identified. In this application, we seek to identify the first natural thymus ligand of a Treg cell. We will identify the thymic ligand of the 2P24 Treg, which is a typical natural Treg cell. We will approach this goal by taking advantage of two properties that the agonist of 2P24 Treg cells has. One, that 2P24 TCR recognizes a ligand expressed in the epidermis, and two, that the expression of the selecting ligand of 2P24 cells in the thymus is dependent on an intact Aire gene. When 2P24 mice were crossed with Foxp3-deficient mice, they developed a severe skin inflammation, and no other detectable pathology. Furthermore, epidermis lysates from normal mice and primary cultured keratinocytes could stimulate 2P24 cells, but lysates from five other tissues could not. Regarding Aire, the number of thymic 2P24 Treg cells was reduced by more than 90% in 2P24 Aire KO mice. We used bioinformatics to define the list of potential gene(s) encoding the 2P24 ligand, thus helping us to prioritize the search. By combining the two properties (epidermal expression and Aire-dependency) we have narrowed the list of candidates to a very manageable 21 genes. We have set up an assay in which epidermal cells lysates, but not lysates from other tissues, stimulate 2P24 immature cells when presented by splenic antigen-presenting cells (APC) or bone marrow-derived APC. The identification of the 2P24 thymic Treg ligand will be developed in two specific aims. In the first aim, we will identify the epidermal gene that stimulates 2P24 T cells, by using a combination of loss of function and gain of function approaches. We will use CRISPR- mediated elimination of specific genes in primary cultured epidermal cells, and, as a gain of function, we will express the candidate genes in cells that normally don't express it, subsequently allowing antigen presentation by APC expressing the selecting MHC allele, and assessing 2P24 T cell reactivity. In the second aim, we will eliminate the gene identified in aim 1 specifically in medullary thymus epithelium, as well as induce 2P24 Treg cell differentiation by intrathymic injection of the agonistic 2P24 peptide. This peptide should not induce Treg cell differentiation of a Treg with different TCR, A12, for example. Overall, this application tests key hypotheses on the limiting agonist model of Treg cell development. Success in the identification of the 2P24 agonist will allow key follow up experiments, for the first time using defined peptides that induce natural Treg cell in the thymus.